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Li Xuan Tan, Gulpreet Kaur, Gurugirijha Rathnasamy, Nilsa LaCunza, Aparna Lakkaraju; Aberrant endosome dynamics in the retinal pigment epithelium promote complement C3 activation in models of macular degeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3992.
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Inherited and age-related macular degenerations are strongly associated with abnormal complement activity. The retinal pigment epithelium (RPE) is the first line of defense against complement attack. We have shown that impaired endo-lysosome trafficking compromises complement-regulatory mechanisms in the RPE. Here, we asked how endosome biogenesis and function regulate the activity of C3, the central player in the complement cascade, in the RPE.
RPE flatmounts and retinal cryosections from pigmented wild-type and Abca4−/− (Stargardt disease) mice were immunostained for endosome markers and complement proteins. C3 activation and cleavage were monitored by immunoblotting of mouse RPE lysates. High-speed spinning disk microscopy was used for live imaging of endosome biogenesis, fusion, and uptake of C3 in primary porcine RPE monolayers.
We observed a dramatic increase in the volume and number of early endosomes in Abca4-/- mouse RPE, which have high levels of vitamin A metabolites similar to patients with Stargardt disease. Our lab has shown that the RPE of these mice also accumulate cholesterol, which activates acid sphingomyelinase (ASMase), leading to increased ceramide levels. Consistent with the ability of ceramide to induce inward budding in membranes, we observed accelerated formation of enlarged early endosomes from the plasma membrane in RPE with vitamin A metabolites. Furthermore, drugs that lower cholesterol or inhibit ASMase restored early endosome size. Early endosomes regulate intracellular complement activity by internalizing and recycling C3, whose cleavage is an initial step of complement activation. Live imaging revealed that RPE with vitamin A metabolites endocytose more C3 from the extracellular milieu compared to healthy RPE. We also observed more C3 within early endosomes and increased cleavage of C3 in RPE from Abca4−/− mice compared to RPE from age-matched wild-type mice, indicative of increased complement activation.
Our findings suggest that early endosome expansion in RPE in models of macular degeneration could aberrantly activate the complement system by increasing the intracellular pool of complement C3. Importantly, our studies help identify drugs that restore membrane dynamics and prevent intracellular complement activation in models of macular degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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